Emerging evidence has linked mutations in myofilament proteins to the development of myocardial dysfunction and genetic cardiomyopathy. We recently have identified two novel missense mutations in human cardiac troponin C at amino acid residues 59 (E59D) and 75 (D75Y) from a patient with idiopathic dilated cardiomyopathy. This is the first identified mutation of troponin C in any human disease. Troponin C is responsible for transmitting the Ca2+-binding signal and triggering the contractile cycle. These missense mutations are located within the Ca2+-binding domain that regulates myocardial contraction, and result in decreased myofilament Ca2+ responsiveness. Based on our preliminary results, we hypothesize that the optimal spatial relationship between helix A and Ca2+-binding loop II must be maintained for proper TnC-regulated Ca2+ signaling in cardiac myofilaments. To test this hypothesis, we propose a systematic, multidisciplinary approach utilizing three integrated levels of investigation: isolated proteins, cardiac myocytes and transgenic animals. First, we will use a mutational model system by generating a number of troponin C mutants based on replacing specific amino acid residues located within regulatory Ca2+-binding domain and define the Ca2+-binding properties of the troponin C mutants (Specific Aim 1). Secondly, we will use adenovirus-mediated gene delivery technique to express specific troponin C mutants in contractile apparatus to elucidate how mutations in cardiac troponin C alter myofilament Ca2+ responsiveness and myocyte contractility (Specific Aim 2). Finally, we will generate a transgenic mouse over-expressing troponin C found in IDCM heart to reveal whether troponin C mutants cause dilated cardiomyopathy (Specific Aim 3). The results obtained from this proposal will help us to understand the physiological role of specific structural alterations in troponin C in the regulation of Ca2+-signaling in cardiac myofilaments in normal and diseased myocardium. Furthermore, the knowledge gained here may contribute to the future development of therapeutic agents using troponin C as a target protein for the treatment of myocardial dysfunction in heart disease.